A genomics-guided approach for discovering and expressing cryptic metabolic pathways

Genome analysis of actinomycetes has revealed the presence of numerous cryptic gene clusters encoding putative natural products. These loci remain dormant until appropriate chemical or physical signals induce their expression. Here we demonstrate the use of a high-throughput genome scanning method to detect and analyze gene clusters involved in natural-product biosynthesis. This method was applied to uncover biosynthetic pathways encoding enediyne antitumor antibiotics in a variety of actinomycetes. Comparative analysis of five biosynthetic loci representative of the major structural classes of enediynes reveals the presence of a conserved cassette of five genes that includes a novel family of polyketide synthase (PKS). The enediyne PKS (PKSE) is proposed to be involved in the formation of the highly reactive chromophore ring structure (or “warhead”) found in all enediynes. Genome scanning analysis indicates that the enediyne warhead cassette is widely dispersed among actinomycetes. We show that selective growth conditions can induce the expression of these loci, suggesting that the range of enediyne natural products may be much greater than previously thought. This technology can be used to increase the scope and diversity of natural-product discovery.

[1]  M. Marahiel,et al.  Crystal structure of the surfactin synthetase‐activating enzyme Sfp: a prototype of the 4′‐phosphopantetheinyl transferase superfamily , 1999, The EMBO journal.

[2]  M. Bibb,et al.  Regulation of Bacterial Antibiotic Production , 2001 .

[3]  B. Lugtenberg.  Faculty Opinions recommendation of Genome sequence of an industrial microorganism Streptomyces avermitilis: deducing the ability of producing secondary metabolites. , 2001 .

[4]  M. Yarmolinsky,et al.  A colorimetric assay of lysogenic induction designed for screening potential carcinogenic and carcinostatic agents. , 1979, Environmental mutagenesis.

[5]  J. Thorson,et al.  The Calicheamicin Gene Cluster and Its Iterative Type I Enediyne PKS , 2002, Science.

[6]  T. Kieser.  Practical streptomyces genetics , 2000 .

[7]  Yoshiyuki Sakaki,et al.  Genome sequence of an industrial microorganism Streptomyces avermitilis: Deducing the ability of producing secondary metabolites , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[8]  M. Linenberger,et al.  Mylotarg: antibody-targeted chemotherapy comes of age , 2001, Current opinion in oncology.

[9]  P. Leadlay,et al.  The biosynthetic gene cluster for the polyketide immunosuppressant rapamycin. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[10]  Wen Liu,et al.  Biosynthesis of the Enediyne Antitumor Antibiotic C-1027 , 2002, Science.

[11]  S. Beyer,et al.  Thestr gene cluster for the biosynthesis of 5′-hydroxystreptomycin inStreptomyces glaucescens GLA.0 (ETH 22794): new operons and evidence for pathway-specific regulation by StrR , 1996, Molecular and General Genetics MGG.

[12]  T. Doyle,et al.  Enediyne antibiotics as antitumor agents , 1995 .

[13]  C. Walsh,et al.  Post-translational modification of polyketide and nonribosomal peptide synthases. , 1997, Current opinion in chemical biology.

[14]  D. Dunaway-Mariano,et al.  The Three-dimensional Structure of 4-Hydroxybenzoyl-CoA Thioesterase from Pseudomonas sp. Strain CBS-3* , 1998, The Journal of Biological Chemistry.

[15]  M. Sternberg,et al.  Enhanced genome annotation using structural profiles in the program 3D-PSSM. , 2000, Journal of molecular biology.

[16]  K. Nicolaou,et al.  The enediyne antibiotics. , 1996, Journal of medicinal chemistry.

[17]  B. Barrell,et al.  Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2) , 2002, Nature.

[18]  P. Liras,et al.  Organization and expression of genes involved in the biosynthesis of antibiotics and other secondary metabolites. , 1989, Annual review of microbiology.